1. Field of the Invention
This invention relates to a novel reagent for assaying hydrogen peroxide wherein peroxidase acts as a catalyst or takes part in the assay and also a method of quantitative assay for hydrogen peroxide.
2. Development of the Invention
Recently, the specificity of an enzymological analytical method in clinical inspection is highly evaluated and the analytical method is rapidly spreading. As the analytical method of measuring urine and glucose, uric acid, cholestrol, triglyceride, lactic acid, creatinine, free fatty acid, glutamylpyruvate transminase, glutamyloxalacetate transaminase, cholinesterase, creatine phosphokinase, lactic acid dehydrogenase, etc., in body fluid, a method of assaying a desired material by assaying hydrogen peroxide formed by reacting an oxidase acting on a material to be finally assayed in each system with the material has been frequently used.
As conventional methods for assaying hydrogen peroxide, there are known a method of converting a chromogen such as o-tolidine, 2,7-diaminofluorenone, N,N-dimethyl-p-phenylenediamine, o-dianisidine, o-aminophenol, triarylimidazoles, etc., into a colored material of an oxidized form and colorimetrically measuring the colored material, a method for oxidative-coupling of a combination of 4-aminoantipyrine and phenol, an N,N-dialkylaniline or an N,N-dialkyltoluidine, a combination of 3-methyl-2-benzothiazolinonehydrazone and o-tolidine, N,N-dimethylaniline or N,N-diethylaniline; or a chromogen such as 4-methoxy-1-naphthol forming a dimer and a derivative thereof, to form a colored material and colorimetrically measuring the colored material, etc. However, in such colorimetric method, there is a limitation on the assaying sensitivity and it is impossible by such conventional colorimetric methods to determine a very small amount of hydrogen peroxide.
Also, as a determination method by a fluorescent method, there is known a method of using homovanillic acid but it is very difficult to determine a very small amount of hydrogen peroxide by such an assay method. For overcoming the difficulty, a method of using fluorescin is proposed as is disclosed in Japanese patent application (OPI) No. 43791/79 (the term "OPI" as used herein refers to a published unexamined Japanese patent application.) but since fluorescin has a hydroquinone-like structure and hence is liable to be oxidized, there is a problem on the stability of the method. As an improvement for the method, a method of using diacetylfluorescin is proposed as is disclosed in Japanese patent application (OPI) No. 48656/80. However, in the case of using the compound, its preferred pH range is 7.5 to 9.5, which differs from the optimum pH for glucose oxidase and cholesterol oxidase. Therefore, the development of reagents which are stable and can be used for the determination of hydrogen peroxide in a low pH region.